Mycophenolic Acid In Human Plasma Research Articles

High-throughput detection of mycophenolic acid in human plasma based on sensitive and rapid fluorescence nitrogen-doped carbon dots sensing platform

In this experiment, a water-soluble, nitrogen-doped yellow-green fluorescent N-doped carbon dots (N-CDs) were synthesized by one-step hydrothermal method using β-cyclodextrin as carbon source and L-phenylalanine as nitrogen source. The fluorescence quantum yield of the obtained N-CDs was as high as 9.96%, and the N-CDs exhibited photostability at different pH, ionic strength and temperature. The morphology of the N-CDs was approximately spherical with an average particle size of about 9.4 nm. Based on the fluorescence enhancement effect of mycophenolic acid (MPA) on N-CDs, a quantitative detection method of MPA was established. This method had good selectivity and high sensitivity for MPA. The fluorescence sensing system was applied to the detection of MPA in human plasma. The linear range of MPA were 0.06–3 μg·mL−1 and 3–27 μg·mL−1 with a detection limit of 0.016 μg·mL−1, and the recoveries were 97.03∼100.64 % with the RSDs of 0.13∼2.90 %. The interference experiment results showed that the interference of other coexisting substances, including Fe3+, can be ignored in the actual detection. Comparing the results measured by the established method with the EMIT method, it was found that the results obtained by the two methods were similar, and the relative error was within ± 5 %. This study provided a simple, rapid, sensitive, selective and effective method for the quantitative analysis of MPA, and was expected to be applied to clinical MPA blood concentration monitoring.

Development and application of a thin-film molecularly imprinted polymer for the measurement of mycophenolic acid in human plasma.

Mycophenolic acid (MPA) is used to suppress the immune response following organ transplantation; however, complex pharmacokinetic behavior and a large interpersonal variability necessitate therapeutic drug monitoring. To overcome the limitations of current sample preparation techniques, we present a novel thin-film molecularly imprinted polymer (TF-MIP) extraction device as part of a simple, sensitive, and fast method for analysis of MPA from human plasma. Mycophenolic acid is extracted from plasma using a tailor-made TF-MIP that is subsequently desorbed into an organic solvent system compatible with mass spectrometry. The MIP yielded higher recovery of MPA relative to a corresponding non-imprinted polymer. The method allows for the determination of MPA in 45 min including analysis time and can be scaled for high throughput to process as many as 96 samples per hour. The method gave an LOD of 0.3 ng mL-1 and was linear from 5 to 250 ng mL-1 . Patient plasma samples (35 μL) were diluted using charcoal-stripped pooled plasma to a final extraction volume of 700 μL; when MPA in patient plasma is high, this ratio can easily be adjusted to ensure samples are within the method linear range. Intra- and inter-day variability were 13.8% and 4.3% (at 15 ng mL-1 ) and 13.5% and 11.0% (at 85 ng mL-1 ), respectively (n=3); inter-device variability was 9.6% (n=10). Low inter-device variability makes these devices suitable for single use in a clinical setting, and the fast and robust method is suitable for therapeutic drug monitoring, where throughput and time-to-result are critical.

Quantification of mycophenolic acid in human plasma by liquid chromatography with time-of-flight mass spectrometry for therapeutic drug monitoring.

This study presents, for the first time, the development and validation of a liquid chromatography and time-of-flight mass-spectrometry (LC-TOF-MS) based assay to quantify mycophenolic acid (MPA) in patient samples as part of a routine therapeutic drug monitoring service. MPA was extracted from 50 μl human plasma by protein precipitation, using sulindac as internal standard (IS). Separation was obtained on a Luna™ Omega polar C18 column kept at 40°C. The mobile phase consisted of a mixture of acetonitrile-deionized water (50:50, v/v) with 0.1% formic acid at a flow rate of 350 μl/min. Analyte and IS were monitored on a TOF-MS using a Jet-Stream™ (electrospray) interface running in positive mode. Assay performance was evaluated by analysing patient plasma (N = 69) and external quality assessment (N = 6) samples. The retention times were 2.66 and 2.18 min for MPA and IS, respectively. The lower limit of quantification of MPA was 0.1 μg/ml. The within- and between-assay reproducibility results ranged from 1.81 to 10.72%. Patient and external quality assessment sample results were comparable with those obtained previously by an in-house validated LC-MS/MS method. This method showed satisfactory analytical performance for the determination of MPA in plasma over the calibration range of 0.1-15.0 μg/ml.

Determination of mycophenolic acid in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its pharmacokinetic application in kidney transplant patients.

To implement and validate an analytical method by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC MS/MS) to quantify mycophenolic acid (MPA) in kidney transplant patients. Quantification of MPA was performed in an ACQUITY UPLC H Class system coupled to a Xevo TQD detector and it was extracted from plasma samples by protein precipitation. The chromatographic separation was achieved through an ACQUITY HSS C18 SB column with 0.1% formic acid and acetonitrile (60:40vol/vol) as mobile phase. The pharmacokinetic parameters were calculated by non-compartmental analysis of MPA plasma concentrations from 10 kidney transplant patients. The linear range for MPA quantification was 0.2-30 mg/L with a limit of detection of 0.07 mg/L; the mean extraction recovery was 99.99%. The mean intra- and inter-day variability were 2.98% and 3.4% with a percentage of deviation of 8.4% and 6.6%, respectively. Mean maximal concentration of 10 mg/L at 1.5 h, area under the concentration-time curve of 36.8 mg·h/L, elimination half-life of 3.9 h, clearance of 0.32 L/h/kg and volume of distribution of 1.65 L/kg were obtained from MPA pharmacokinetics profiles. A simple, fast and reliable UPLC-MS/MS method to quantify MPA in plasma was validated and has been applied for pharmacokinetic analysis in kidney transplant patients.

Validation of ultra performance liquid chromatography photodiode array method for determination of mycophenolic acid in human plasma and its application

Validation of ultra performance liquid chromatography photodiode array method for determination of mycophenolic acid in human plasma and its application

Handling of highly coeluted chromatographic peaks by multivariate curve resolution for a complex bioanalytical problem: Quantitation of selected corticosteroids and mycophenolic acid in human plasma

The present study describes the analytical performance of a fast-elution protocol and smart methodology based on multivariate curve resolution-alternating least square (MCR-ALS) modeling of high performance liquid chromatography with photodiode-array detection (HPLC-DAD) data for simultaneous determination of prednisolone (Predl), methylprednisolone (Mpredl) and mycophenolic acid (MPA) in plasma samples. The LC method optimized at two isocratic reverse phase over a symmetric C18 column, a 60:40 (v/v) mixture of acetonitrile and water (0.02 M KH2PO4 (pH = 3.7) buffer solution) and a 10:70:20 (v/v/v) mixture of acetonitrile, methanol and water. The most challenges in the present study were the severe coelution of analytes of interest with each other and the matrix interferences, and high spectral similarity of selected corticosteroids, depending on the mobile phase method. To circumvent these drawbacks, the whole chromatographic runs were divided into two sections. Then, the matrix augmentation in spectra and retention time direction were implemented for the first and second regions, respectively. Highly acceptable resolution and quantification results were obtained. The average recoveries were 97.3% and 102.1% for Predl, 96% and 98.6% for Mpredl and 97.7% and 100.8% for MPA, using two mobile phase conditions, respectively. Accurate and precise results, elimination of expensive and time consuming sample pretreatment steps and a very short chromatographic run time, are among the advantages of the presented method.

A novel and Rapid LC–MS/MS assay for the Determination of Mycophenolate and Mycophenolic Acid in Human Plasma

Objective : The main goal of this research was to develop a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/ MS) method for the simultaneous quantification of mycophenolate mofetil and mycophenolic acid. Methods: Sample extraction was carried out using a simple solid phase extraction (SPE) technique. The extracted samples were chromatographed on a C 18 column using an isocratic mobile phase composed of acetonitrile and 0.1% formic acid buffer (80:20, v/v) pumped at a flow rate of 1.00 mL/min. Results : Method linearity was established in the concentration range of 0.10–20.0 ng/mL for mycophenolate mofetil and 101–19955 ng/mL for mycophenolic acid. Intra–day and inter–day precision and accuracy results of mycophenolate mofetil and mycophenolic acid were well within the acceptance criteria specified in the US FDA and EMEA guidelines. Conclusion: The projected LC–MS/MS assay method is simple, rapid and sensitive for the simultaneous determination of mycophenolate mofetil and mycophenolic acid in human plasma. This method was successfully used to quantitate the in–vivo plasma concentrations obtained from a pharmacokinetic study. Key words: Mycophenolate mofetil, Mycophenolic acid, Human plasma, Liquid chromatography–tandem mass spectrometry (LC–MS/MS), Method validation, Pharmacokinetics.

Accurate Method of HPLC-Ms/Ms Determination of Mycophenolic Acid in Human Plasma

A simple, accurate and precise method was developed using HPLC-MS/MS to quantify mycophenolic acid in human plasma. Mycophenolic acid-D3 was used as internal standard. Sample preparation involved protein precipitation. Chromatographic analysis was performed on a Phenomenex Kinetex C18 (30 mm × 4.6 mm, 2.6 μm) column using acetonitrile-water as the mobile phase. Detection in negative ionization mode was used to quantify mycophenolic acid by multiple reaction monitoring. The calibration curve was linear in the range of 0.5-30 μg/mL, with intra-and inter-batch values for accuracy and precision (coefficient of variation) ranging from 99.76 to 111.38% and from 2.54 to 9.01%, respectively. The method was successfully applied to conduct a pharmacokinetic study of 360 mg coated tablet Myfortic in 48 healthy participants; 768 blood samples were analysed.

Determination of mycophenolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry

A simple, sensitive and high throughput ultra performance liquid chromatography tandem mass spectrometry method has been developed for the determination of mycophenolic acid in human plasma. The method involved simple protein precipitation of MPA along with its deuterated analog as an internal standard (IS) from 50µL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 (100mm×2.1mm, 1.7µm) column under isocratic conditions using acetonitrile and 10mM ammonium formate, pH 3.00 (75:25, v/v) as the mobile phase. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for quantitation. In-source conversion of mycophenolic glucuronide metabolite to the parent drug was selectively controlled by suitable optimization of cone voltage, cone gas flow and desolvation temperature. The method was validated over a wide concentration range of 15–15000ng/mL. The mean extraction recovery for the analyte and IS was >95%. Matrix effect expressed as matrix factors ranged from 0.97 to 1.02. The method was successfully applied to support a bioequivalence study of 500mg mycophenolate mofetil tablet in 72 healthy subjects.

Analytical Validation of a Homogeneous Immunoassay for Determination of Mycophenolic Acid in Human Plasma

Mycophenolic acid (MPA) is an immunosuppression agent for the prophylaxis of organ rejection in patients receiving allogeneic transplants. The drug is administered based in 2 formulations, mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS). MPA acts by specific, reversible, uncompetitive inhibition of inosine monophosphate dehydrogenase (IMPDH) and thus blocks the proliferation of both T- and B-activated lymphocytes. Therapeutic drug monitoring (TDM) constitutes an important part of immunosuppressive treatment because of the demonstrated significant intraindividual and interindividual variability of its pharmacokinetic behavior. TDM is required to optimize immunosuppressive efficacy. We present the analytical validation of a homogeneous particle-enhanced turbidimetric inhibition immunoassay (PETINIA) technique for determination of MPA in human plasma, and compare with a homogeneous enzyme immunoassay technique (EMIT; reference method), both methods adapted on a Dimension analyzer (Siemens). We examined 50 human plasma samples from kidney transplant recipients treated with MMF or EC-MPA, which were analyzed simultaneously by both methods. The interassay precision was 5.95% at a concentration of 1.0 μg/mL, 3.47% at 7.5 μg/mL, and 3.75% at 12.0 μg/mL. The bias of PETINIA-MPA for each of the 3 quality control sample was <3.0%. Least squares linear regression yielded an r-value of 0.994 with the following linear regression equation: PETINIA = 0.939 * EMIT − 0.063. Bland-Altman comparison presented a mean negative difference of −0.312 μg/mL (standard deviation [SD], 0.441), namely, −7.6% for PETINIA-MPA. The PETINIA assay for monitoring MPA concentrations is an acceptable method for routine clinical use, with interassay imprecision (% coefficient of variation) ranging from 5.9% to 3.7% below and above the therapeutic concentration range, respectively. In conclusion, MPA-EMIT and PETINIA-MPA methods on Dimension analyzer have a good correlation (r = 0.994), but PETINIA-MPA method demonstrates a negative average difference of −7.6% in comparison with EMIT-MPA method.

High-performance Liquid Chromatography Method for the Determination of Mycophenolic Acid in Human Plasma and Application to a Pharmacokinetic Study of Mycophenolic Acid Dispersible Tablet

A sensitive and selective high-performance liquid chromatographic-ultraviolet (HPLC-UV) method for the determination of mycophenolic acid (MPA, CAS 24280-93-1) in human plasma has been developed. Sample treatment was based on protein precipitation with a trichloroacetic acid-water (10:90, w/v) solution. The analytical determination was carried out by HPLC with ultraviolet detection at 254 nm. Chromatographic separation was achieved on a C18 column by isocratic elution with acetonitrile-water (pH 4.4) (50:50, v/v) at a flow rate of 1.0 mL/min. The method was linear in the concentration range of 0.2-50.0 microg/mL. The lower limit of quantification (LLOQ) was 0.2 microg/ mL. The intra-day and inter-day relative standard deviations across three validation runs over the entire concentration range were less than 7.05%. The accuracy determined at three concentrations (0.4, 5.0 and 20.0 microg/mL for MPA) was within +/- 10.0%. The method was successfully applied to the evaluation of the pharmacokinetic profile of MPA dispersible tablet in 20 healthy volunteers. The results showed that AUC, C(max) and T1/2 for the test and reference formulations were not significantly different (P > 0.05). The relative bioavailability was 96.42 +/- 15.5%.

Bioequivalence and pharmacokinetic comparison of two mycophenolate mofetil formulations in healthy Chinese male volunteers: An open-label, randomized-sequence, single-dose, two-way crossover study

Background: Mycophenolate mofetil (MMF) is an ester prodrug of mycophenolic acid (MPA), so clinical studies measure the circulating plasma MPA concentration instead of MMF. MPA is extensively glucuronidated by several uridine diphosphate glycosyltransferases into an inactive 7-O-glucuronide and a pharmacologically active acylglucuronide. Considering the effect of racial differences and genetic factors on the pharmacokinetic (PK) properties of drugs, it is necessary to study them in Chinese populations. Objectives: The aim of this study was to compare the clinical bioequivalence and PK properties of a test (dispersible tablets) and reference (capsules) formulation of MMF 1.0 g in healthy Chinese volunteers. We also established a validated HPLC method for the determination and quantification of MPA in human plasma. The study was required to obtain Chinese regulatory approval for the test formulation. Methods: This open-label, randomized-sequence, single-dose, 2-way crossover study was conducted at the First Hospital of Nanjing Medical University, Nanjing, China. Eligible subjects were healthy male volunteers who were randomly assigned at a 1:1 ratio to receive a single 1.0-g dose of the test or reference formulation, followed by a 1-week washout period and administration of the alternate formulation. The plasma concentration of MPA, which is the active metabolite of MMF, was determined using a validated HPLC method. For analysis of PK properties, blood samples were collected at 0, 10, 20, 30, and 45 minutes, and 1, 1.5, 3, 5, 8, 11, 18, 36, and 48 hour(s). The PK parameters, including C max, T max, t ½, AUC 0–48, and AUC 0–∞, were determined from the plasma concentrations of the 2 formulations by noncompartmental analysis. Tolerability was assessed at baseline (be- fore administration) and at 30 minutes and 1, 5, 18, and 48 hours after administration by monitoring vital signs. Laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis) were performed for the identification of adverse events (AEs) (eg, leukopenia, thrombocytopenia, anemia). Patient interviews were conducted to assess the occurrence of AEs such as diarrhea, abdominal pain, nausea, vomiting, and secondary infections. The formulations were considered to meet the regulatory requirements of bioequivalence if the log-transformed ratios of C max and AUC were within the predetermined equivalence range (80%-125%) as established by the US Food and Drug Administration (FDA). Results: Eighteen healthy Chinese male volunteers (mean [range] age, 23.5 [22–30] years; weight, 63.3 [56–68] kg; height, 171 [165–184] cm) were enrolled and completed the study. The main PK parameters of the MMF test and reference formulations were as follows: mean (SD) T max, 0.68 (0.21) and 0.81 (0.18) hour, respectively; C max, 25.58 (4.79) and 26.47 (3.67) mg/L; AUC 0–48, 59.19 (9.23) and 58.32 (9.28) mg/L/h; t ½, 15.12 (3.17) and 16.04 (4.22) hours; AUC 0–∞), 63.28 (10.23) and 62.41 (10.28) mg/L/h. The mean (SD) relative bioavailability was 101.5% (10.3%). No statistically significant differences were found based on ANOVA. The ratios of C max (0.97) and AUC (1.01) for the test and reference formulations were within the FDA bioequivalence definition intervals of 80% to 125%. No AEs were reported by subjects or found on analysis of vital signs or laboratory tests. Conclusions: In this study in healthy Chinese male volunteers, results from the PK analysis suggested that a single dose of the test and reference formulations of MMF 1.0 g met the regulatory requirements of bioequivalence, based on the FDA regulatory definition (rate and extent of absorption). Both formulations were well tolerated.

Development of a CE method for the determination of mycophenolic acid in human plasma: A comparison with HPLC

Mycophenolate mofetil (MMF) is a widely used drug for the maintenance of immunosuppressive therapy in renal-transplant recipients. MMF is rapidly metabolized in vivo to mycophenolic acid (MPA), a reversible, noncompetitive inhibitor of inosine monophosphate dehydrogenase, which represents a limiting enzyme in lymphocyte proliferation. MPA shows large interindividual pharmacokinetic variability: its monitoring is therefore of primary importance to achieve adequate immunosuppression with minimized risk of graft rejection or toxicity. We developed a CE method for the determination of total MPA (tMPA) in plasma, based on easy sample preparation; CE evaluation of tMPA was performed in 30 mmol/L sodium-borate with 10 mmol/L SDS (pH 10.00) at 25 degrees C using a 60 cm (54.5 to window) uncoated capillary with UV detection at 254 nm wavelength. MPA was readily detectable in plasma; the CE method was linear in the range of 0.7-120 microg/mL (r >0.992). Intra- and interassay imprecision was <7% except for the lowest spiked MPA concentration, which had an intra-assay RSD% of 14.7 compared to 18.3 interassay. Data by CE were compared with results obtained by a validated HPLC method. CE assay of tMPA exhibited a very good correlation (r(2) >0.988) with respect to HPLC; Bland-Altman difference versus average showed a mean of -0.18 microg/mL +/- 1.14 SD. CE determination of tMPA is a robust, sensitive and reproducible method with the advantage over HPLC of being fast, simple and unexpensive, also enabling quick assessment of MPA for pharmacokinetic studies.

Simple high performance liquid chromatographic assay for mycophenolic acid in renal transplant patients

A selective and highly accurate HPLC-UV method is described to determine plasma concentrations of mycophenolic acid (MPA), the active metabolite of the prodrugs Cellcept ® and Myfortic ®. The method is simple and utilizes acidification of plasma and protein precipitation step using a mixture of acetonitrile and phosphate buffer (pH 3). Following vortex mixing and centrifugation, the supernatant (50 μL) was injected onto a Zorbax Eclipse XDB C 18 column (150 mm × 4.6 mm I.D., 5 μm particle size). A mobile phase composed of acetonitrile and 0.1 M phosphate buffer, pH 3 (43:57) delivered at 1.0 mL/min produced peaks for MPA and the internal standard (Naproxen) in <7 min. Calibration curves were linear ( r 2 > 0.994) from 1.0–40 μg/mL with intra- and inter-day precision <15% and accuracy >95%. The method's improved sensitivity (LOQ = 1.0 μg/mL) and minimal sample processing allowed rapid monitoring of MPA in human plasma.

Molecularly imprinted solid-phase extraction for rapid screening of mycophenolic acid in human plasma

A molecularly imprinted solid-phase extraction coupled with high performance liquid chromatography (MISPE–HPLC) method was developed for rapid screening of mycophenolic acid (MPA) in human plasma. MPA imprinted polymers (MPA-MIP) were synthesized and then tested for their performance both in organic and in aqueous solution. MPA was selectively trapped and preconcentrated on the MPA-MIP sorbent using different loading and washing conditions. The good selectivity of MPA-MIP enabled further clean-up of the interfering components in human plasma. For the proposed MISPE–HPLC method, the linearity between responses (peak area) and concentration was found over the range of 1–100 μg/ml with a linear regression coefficient ( R 2) of 0.9989. The limit of detection (LOD) and theoretical limit of quantification (LOQ) for MPA in plasma were 0.10 and 0.32 μg/ml, respectively. The precisions were 7.3, 3.5 and 4.7% RSD for intra-day assay and 9.2, 4.1 and 5.5% RSD for inter-day reproducibility, respectively, at three concentration levels of MPA in spiked plasma (1, 10 and 100 μg/ml). Both recoveries for the extraction (more than 74%) and for the analytical method (more than 87%) were acceptable for screening MPA in plasma samples. Twelve-hour pharmacokinetic profile of MPA for a renal transplant recipient receiving chronic oral dosing of 500 mg mycophenolate mofetil (MMF) was investigated. Results indicated that this method could be applied for therapeutic drug monitoring of mycophenolic acid in patient plasma.

Quantification of total and free mycophenolic acid in human plasma by liquid chromatography with fluorescence detection

A simple high-performance liquid chromatographic (HPLC) method was developed for the assay of total and free mycophenolic acid (MPA) in human plasma. Prior to analysis, total mycophenolic acid was extracted by protein precipitation and free drug was isolated from plasma samples using ultrafiltration. The extracts were injected onto a Kromasil C 8 column at 30 °C with excitation and emission wavelengths set at 342 and 425 nm, respectively. The mobile phase was consisted of acetonitrile-32 mM glycine buffer, pH 9.2 (20:80, v/v), at a flow rate of 1.0 ml/min. The method was found to be linear over the concentration range investigated, 0.05–40 mg/l for total mycophenolic acid ( r > 0.999) and 5–1000 μg/l ( r > 0.99) for free drug. The percentage error of the analytical method was below 10.9%. The intra- and inter-day reproducibility was adequate with the coefficients of variation of 8.28% or below. The run time were 4 and 6 min for free and total MPA, respectively. The method thus can be effectively applied to measure mycophenolic acid concentrations in clinical samples.

Determination of mycophenolic acid in human plasma by high-performance liquid chromatography

The development, validation and evaluation of high-performance liquid chromatography (HPLC) method for quantifying mycophenolic acid in human plasma is described. The method involved protein precipitation using acetonitrile, after addition of terazosin as an internal standard. Separation was achieved with a reversed-phase C 18 column ( 250 mm×4.6 mm) employing UV detection at 215 nm. The mobile phase consisted of 0.02 M potassium dihydrogenphosphate solution adjusted to pH 6.9 with 2 M potassium hydroxide solution–acetonitrile (80:20 (v/v)) at a flow rate of 1.5 ml/min. The total run time was 21.0 min. The assay was linear from 0.2 to 25 μg/ml with goodness of fit ( r 2) greater than 0.99 observed with three precision and accuracy batches during validation. The observed mean recoveries were 89.3 and 98.0% for drug and internal standard, respectively. The applicability of this method to pharmacokinetic studies was established after successful application during a 34-subject bioavailability study. The method was found to be precise, accurate and specific during the study.

Liquid chromatographic assay for simultaneous determination of abacavir and mycophenolic acid in human plasma using dual spectrophotometric detection

A combined bio-analytical assay for abacavir, a reversed transcriptase inhibitor, and mycophenolic acid (MPA), based on reversed-phase liquid chromatography and both ultraviolet (UV) absorption and fluorescence detection, is reported. Both analytes are extracted from plasma with acetonitrile. After centrifugation, evaporation of the supernatant and reconstitution in water, the sample is injected into the chromatograph. Abacavir is detected using UV detection at 285 nm and MPA spectrofluorometrically at 345 and 430 nm for excitation and emission, respectively. The method has been validated in the 80–2000 ng/ml range for abacavir and in the 10–10 000 ng/ml range for MPA for 200-μl plasma samples. The lower limits of quantification are 80 and 10 ng/ml for abacavir and MPA, respectively. Precisions and accuracies are ≤8% in the valid concentration ranges of both analytes.

Determination of the Acyl Glucuronide Metabolite of Mycophenolic Acid in Human Plasma by HPLC and Emit

The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated. The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents. The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2-25 mg/L), <4.4% for 7-O-MPAG (10-250 mg/L), and < or =14% for AcMPAG (0.1-5 mg/L). The between-day imprecision was <6.2%, <4.5%, and < or =14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at -20 degrees C. The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.

Determination of mycophenolic acid in human plasma by HPLC: [formula omitted], Paruszewski R. Department of Drug Chemistry, Medical Academy, 1 Banacha Str., 02-097 Warsaw, Poland

AIM To establish a method for determination of mycophenolic acid in human plasma.METHODS Mycophenolic acid was determined by HPLC using a HYPERSIL C18(150 mm×5 mm,5 μm) column and a guard column ODS(5 mm×3.9 mm,10 μm),the mixture of methanoland and NaH2PO4(pH=3.25) and acetonitrile(40∶40∶20,V/V) as mobile phase with a flow rate of 0.8 mL·min-1.The temperature of column was 25℃ and the wavelength of detection was 254 nm.RESULTS The standard curve was linear in the range of 0.23-46.13 mg·L-1(r=0.999 7),the method recovery was more than 96%,and the intra-day and inter-day RSD were all less than 7%.CONCLUSION This method is reproducible,convenient and sensitive for determination of plasma concentration and can be used to study clinical pharmacokinetic mycophenolic acid.